Rapid detection of antigen-specific mouse IgE by enzyme-linked immunosorbent assay (ELISA).

A rapid, sensitive, antigen-specific mouse IgE capture ELISA is described. A monoclonal rat anti-mouse IgE was used as the capture antibody, and a DNP-coupled BSA-biotinylated conjugate along with a peroxidase-avidin-biotin complex was utilized as the detection system. The lower detection limit of this assay is 8.5 ng/ml of antigen-specific IgE. With some modifications, this assay can be employed to screen for antigen specific antibodies of other isotypes and subtypes.

Estimation of IgE antibody with a nonisotopic technique with no interference of isotypic antibodies.

A modified ELISA was developed to estimate antigen-specific IgE antibody. IgE against ragweed antigen E (AgE) was the model system, and paired pre- and postimmunotherapy sera were evaluated because the latter had been demonstrated to contain significant amounts of IgG antibody. Patients' serum IgE was bound to the surface of wells of a polystyrene microtiter plate previously coated with monoclonal murine antihuman IgE. After washing, AgE conjugated with alkaline phosphatase was added and was specifically bound by IgE. Optical density was recorded after addition of enzyme substrate to the wells and was proportional to the concentration of IgE directed against ragweed AgE in the serum samples. In contrast to many other assay techniques for antigen-specific IgE, interference by specific antibody of other classes cannot occur with this method. Results correlated well with those of a previously described solid-phase radioimmunoassay that required myeloma IgE.

Evaluation of an enzyme immunoassay for total IgE on a semi-automated analyser. A multicentre study.

Total serum IgE was measured by a semi-automated enzyme immunoassay on sera from normal and disease groups. Data from this investigation was analyzed in respect of precision, linearity, sensitivity and correlation with other test methods. Using human serum pools having values between 7 and 480 IU/ml, the intra-assay coefficient of variation ranged from 3.3 to 14.6% with an arithmetic mean of 6%. The inter-assay coefficient of variation on commercially supplied control sera ranged from 4.4 to 14.2%. In addition, tests were carried out on serially diluted samples to assess the linearity of the method, and on sera with IgE levels of less than 5 IU/ml in order to assess its sensitivity. It was shown that the technique being assessed was unaffected by the presence of lipid or haemoglobin or by the addition of bilirubin or any one of 46 commonly prescribed drugs each at double its toxic dose. There was good correlation between the semi-automated enzyme immunoassay technique and four other methods used during this study. This technique exhibits excellent specificity, reproducibility and a sensitivity well within clinical demands.

[ImmunoComb--reliability and reproducibility]. / ImmunoComb fiabilité et reproductibilité.

ImmunoComb is a solid phase in vitro immunological test for the measurement of serum or plasma total IgE. The technique was compared with PRIST for measurement of total IgE. The study also included reproducibility, on a test serum dilution both on a complete plate and plate sections. Statistically the conclusion is that ImmunoComb is a colourimetric test that gives the exact total IgE titre with good reproducibility. Use of a photometer greatly improves the sensitivity of assessing the results.

[Total serum IgE: normal pediatric values using the FAST IgE fluoroimmunoenzymatic technic]. / IgE totales sériques: valeurs usuelles en pédiatrie par la technique fluoro-immuno-enzymatique IgE FAST.

This reports covers the application to paediatrics of total IgE determination by the means of a new direct, sandwich type, fluorescence enzyme immuno-assay (Fluoro-allergo sorbent test:FAST IgE). The technical process is adapted for usual determinations, in case of infants and young children, of low normal rates with a good sensitivity, and of high pathological rates. IgE total usual values are established by the FAST IgE test from serums of healthy children, aged from one day to 14 years. Seven age groups are to be distinguished.

Variability of IgE protein measurement in cell-culture supernatants: results from a multicenter collaborative study.

The sensitivity, specificity, and precision of immunoassays for quantitation of IgE in cell-culture supernatants were tested in a multicenter trial involving 22 laboratories. Fourteen coded test samples included cell-culture medium alone, culture medium with varying concentrations of polyclonal or myeloma IgE, and medium from unstimulated or pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cell (PBMC)-culture supernatants. Two laboratories reported measurable IgE in non-IgE-containing control samples. Although the IgE content of a single 0.50 ng/ml polyclonal IgE sample should have been measured easily by the claims of all laboratories, only 13 laboratories measured IgE in this sample in 22 of the 48 assays. Most laboratories could measure both polyclonal and myeloma IgE at 5.0 ng/ml; however, the IgE determinations for the myeloma proteins were nearer the predicted value. Although 15 laboratories found measurable IgE in the PWM-stimulated PBMC-culture supernatants from a single nonatopic donor, the levels did not differ significantly from that measured in unstimulated PBMC-culture supernatants. Three laboratories reported considerably higher IgE levels in the PWM-stimulated PBMC-culture supernatants than in other PBMC-culture supernatants. Only 13 laboratories could quantitate IgE in each of coded duplicate samples containing 0.50 ng/ml polyclonal IgE. These findings indicate a wide variability in the sensitivity, specificity, and precision of assays used to quantitate low levels of IgE protein. Investigators should be encouraged to make greater use of existing national and international reference materials in the standardization and performance of their IgE immunoassays.

A comparison of the Phadebas and Phadezym IgE paper immunosorbent test kits.

The Phadebas (isotype label) and Phadezym (enzyme label) paper immunosorbent test kits have been compared. Precision and ease of operation are shown to be comparable for both kits, and the advantages and disadvantages of each are similar. The enzymatic labelled kit seems to provide a suitable alternative for laboratories wishing to measure IgE but who do not have isotope-counting facilities available.

The Australian reference preparation of human serum immunoglobulins.

An Australian Reference Preparation of human serum immunoglobulins was prepared from pooled sera of 240 healthy, adult donors. The preparation was calibrated for IgG, IgA and IgM levels by radial immunodiffusion against the 1st International Reference Preparation of Human Serum Immunoglobulins G, A and M (IgG, IgA and IgM) and for IgE by PRIST against the 1st International Reference Preparation of Human Serum Immunoglobulin E (IgE). The following potency values were assigned to the Australian Reference Preparation designated ASPS 78-1 (Lyoph.): 100 IU/vial for IgG and IgE, 102 IU/vial for IgA and 114 IU/vial for IgM.